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目的探讨p38丝裂原活化蛋白激酶磷酸化(P-MAPK)水平及半胱氨酸蛋白酶-3(Caspase-3)在N-甲基-D-天冬氨酸(NMDA)诱导的体外培养神经元中的激活及p38MAPK抑制剂SB203580的保护作用。方法随机将原代培养7 d的大鼠皮质神经元分为对照组,NMDA组,SB203580低、中、高剂量组。对照组仅用DMEM/F12完全培养基,NMDA组去除正常神经元培养液,加入50μmol/L NMDA,处理时间10 min;SB203580低、中、高剂量组浓度分别为5、10、20μmol/L,继续培养24 h,加入50μmol/L NMDA。采用MTT法评价细胞生存力,丫啶橙(AO)染色检测凋亡细胞数量及乳酸脱氢酶(LDH)含量;免疫细胞化学染色法(IHC)及免疫印记法(WB)检测皮质神经元内P-MAPK及Caspase-3的表达水平。结果与对照组比较,NMDA组的吸光度值明显降低、神经元凋亡明显增加、P-MAPK及Caspase-3表达增加(P均<0.01),SB203580低、中、高剂量组P-MAPK及Caspase-3随着浓度的增加表达减少,以高剂量组为明显(P<0.05,P<0.01);与NMDA组比较,SB203580低、中、高剂量组吸光度值均升高,以高剂量组为明显,细胞凋亡数量明显减少(P均<0.05)。结论 P-MAPK及Caspase-3在NMDA诱导的皮质神经元中表达增强,SB203580对NMDA诱导的神经元损伤具有保护作用,其作用机制可能与p38MAPK的活化,进而直接或间接激活Caspase-3的表达有关。
Objective To investigate the effects of p38 mitogen-activated protein kinase (P-MAPK) and caspase-3 on N-methyl-D-aspartate Meta-activation and p38MAPK inhibitor SB203580. Methods Primary cortical neurons cultured for 7 days were randomly divided into control group, NMDA group and SB203580 low, medium and high dose groups. The control group received only DMEM / F12 complete medium. The normal neuron culture medium was removed in NMDA group and 50 μmol / L NMDA was added for 10 min. The concentrations of SB203580 low, medium and high dose groups were 5, 10 and 20 μmol / L, Continue to train for 24 h, add 50μmol / L NMDA. Cell viability was assessed by MTT assay. The number of apoptotic cells and the content of lactate dehydrogenase (LDH) were detected by acridine orange (AO) staining. Immunocytochemical staining (IHC) and immunoblotting (WB) P-MAPK and Caspase-3 expression levels. Results Compared with the control group, the absorbance of NMDA group was significantly decreased, the apoptosis of neurons was significantly increased, the expressions of P-MAPK and Caspase-3 were increased (P <0.01), while the expressions of P-MAPK and Caspase -3 decreased with the increase of the concentration, the high dose group was significantly (P <0.05, P <0.01); Compared with the NMDA group, SB203580 low, medium and high dose group were increased in absorbance values to high-dose group was Obviously, the number of apoptotic cells decreased significantly (all P <0.05). CONCLUSIONS: P-MAPK and Caspase-3 are up-regulated in NMDA-induced cortical neurons. SB203580 has a protective effect on NMDA-induced neuronal injury. Its mechanism may be related to the activation of p38MAPK, which in turn directly or indirectly activates the expression of Caspase-3 related.